Structure-based mutation analysis Gaucher Disease

The aim of this task was to carry out a thorough analysis of ten mutations and to classify them as disease-causing and non-disease causing. Technical details are reported in our protocol.

Cystral structure

<figtable id="tab:mutations">

PDB	Res [Å]	R value	Coverage	pH
2nt0	1.80	0.18	96% (40-536)	4.5
3gxi	1.84	0.19	96% (40-536)	5.5
2v3f	1.95	0.15	96% (40-536)	6.5
2v3d	1.96	0.16	96% (40-536)	6.5
1ogs	2.00	0.18	96% (40-536)	4.6

The 5 crystral structures of glycosylceramidase with the highest resolution. The physiological lysosomal pH value is 4.5. 2nt0 was selected for the analysis. </figtable>

Mutations

<figtable id="tab:mutations">

Nr	Pos P04062	Pos 2nt0_A	From	To	Disease causing
1	99	60	H	R	No
2	211	172	V	I	No
3	150	111	E	K	Yes
4	236	197	L	P	Yes
5	248	209	W	R	Yes
6	509	470	L	P	No
7	351	312	W	C	Yes
8	423	384	A	D	Yes
9	482	443	D	N	No
10	83	44	R	S	No

Mutations used for the structure-based mutation analysis. </figtable>

<figure id="fig:mutations">

2nt0_A with the selected mutations used for the structure-based analysis. Blue: wildtype residues; Red: mutant residues; Orange: active site residues E235 and E340.

</figure>

SCWRL

We employed SCWRL <ref name="scwrl">Qiang Wang, Adrian A. Canutescu, and Roland L. Dunbrack, Jr.(2008). SCWRL and MolIDE: Computer programs for side-chain conformation prediction and homology modeling. Nat Protoc.</ref> for substituting the wildtype residues listed in <xr id="tab:mutations"/> by the corresponding mutatant residues which are chosen from a rotamer library. <xr id="fig:scwrl"/> denotes the results.

<figure id="fig:scwrl">

• 

  1: H60R (2.88)

• 

  2: V172I (4.39)

• 

  3: E111K (8.14)

• 

  4: L197P (3.28)

• 

  5: W209R (9.23)

• 

  6: L470P (28.84)

• 

  7: W312C (0.18)

• 

  8: A384D (13.50)

• 

  9: D443N (2.36)

• 

  10: R44S (4.85)

Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; In brackets: energy(mutant)-energy(wildtype). </figure>

None of rotamers chosen by SCWRL clashed with another side-chain or the backbone. The only mutation which led to a structural change was L470P. Here, the insertion of proline interrupted the beta-sheet. The hydrogen bonding network changed in case of mutation number 1, 5, 7, and 8 (cf. <xr id="tab:scwrl"/>). W209R introduces a hydrophilic arginine which forms a hydrogen bond to T180. Although not predicted by SCWRL, the arginine might impact the protein structure. W312C is located next to the active site (cf. <xr id="fig:mutations"/>) and there exists a hydrogen bond to E340. Substitution the hydrophobic tryptohphane by a hydrophlic cysteine in the vicinity of the active site might account for the disease-causing effect of this mutation.

As expected, all mutations increased the energy of the model (cf. the energy difference in brackets in <xr id="fig:scwrl"/>). The energy increased most in case of L470P due to the break of the beta-sheet. A384D and W209R also made the model less stable which is caused by substituting an unpolar residue by a charged residue. All four mutations which increased the model energy most are disease-causing.

<figtable id="tab:scwrl">

Nr	Mutation	Wildtype		Mutatant		Clashes	Structural change
		H-bonds	Hydrophobicity	H-bonds	Hydrophobicity
1	H60R	T471	Hydrophilic	G62	Hydrophilic	No	No
2	V172I		Hydrophobic		Hydrophobic	No	No
3	E111K		Hydrophilic		Hydrophilic	No	No
4	L197P		Hydrophobic		Hydrophobic	No	No
5	W209R		Hydrophobic	T180	Hydrophilic	No	No
6	L470P	T482	Hydrophobic	T482	Hydrophobic	No	Yes
7	W312C	E340, C342, P316	Hydrophobic	E340, C342	Hydrophilic	No	No
8	A384D		Hydrophobic	V404	Hydrophilic	No	No
9	D443N		Hydrophilic		Hydrophilic	No	No
10	R44S	S13, Y487	Hydrophilic	S13, Y487	Hydrophilic	No	No

Structure-based analysis of SNPs from <xr id="tab:mutations"/>. H-bonds: residues involved in forming hydrogen bonds (cut-off: 3.2 Å). </figtable>

We further noticed that SCRWL changed the backbone at some positions which led to different secondary structure assignments (<xr id="fig:scwrl_ss"/>). The positions at which the deviations could be observed were independent from the mutated sites.

<figure id="fig:scwrl_ss">

Seconary structure elements of 2nt0_A (grey) compared to secondary structure elements of models built by SCRWL.

</figure>

FoldX

The superposition of the rotamer configurations predicted by FoldX and SCWRL are shown in <xr id="fig:foldx"/>. The predictions of both tools differed in case of four mutations. In case of H60R, the side-chain orientation of arginine predicted by FoldX forms two instead one hydrogen bonds to T741 and might therefore impact the protein structure more than the orientation of SCRWL. In case of A384D, the romater of FoldX might be more stable than the one of SCWRL since it has a higher distance to the surrounding residues. In case of D443N we prefer the prediction of SCWRL which is closer to the wildtype configuration. For the same reason we prefer the prediction of FoldX in case of R442. For the subsequent GROMACS analysis, we hence chose the FoldX model in case of mutation number 8 and 10 and the SCWRL models for all all other mutations.

<figure id="fig:foldx">

• 

  1: H60R (-0.39)

• 

  2: V172I (-0.80)

• 

  3: E111K (-0.36)

• 

  4: L197P (0.28)

• 

  5: W209R (2.86)

• 

  6: L470P (8.59)

• 

  7: W312C (1.88)

• 

  8: A384D (4.52)

• 

  9: D443N (-0.34)

• 

  10: R44S (0.60)

Rotamers of SNPs from <xr id="tab:mutations"/>. Blue: wildtype; Red: rotamer SCWRL; Orange: rotamer FoldX; In brackets: energy(mutant)-energy(wildtype). </figure>

A comprehensive list of the differences between the mutant and the wildtype models can be found here. The total energy increased in case of mutation number 4-8, and 10. Just as in case of SCWRL (cf. <xr id="fig:scwrl"/>), L470P, A384D, and W209R increased the energy of the model most. Since it is unlikely that mutations like V172I decrease the energy, we consider the energy calculations of SCWRL as more plausible.

Minimise

</figure> </figure>

Energy of the SCWRL models vs. the number of minimise iterations.

Energy of the FoldX models vs. the number of minimise iterations.

<figure id="fig:minmise_scwrl_energies">

<figure id="fig:minmise_foldx_energies">

<figure id="fig:minimise_scwrl_mutations">

• 

  1: H60R

• 

  2: V172I

• 

  3: E111K

• 

  4: L197P

• 

  5: W209R

• 

  6: L470P

• 

  7: W312C

• 

  8: A384D

• 

  9: D443N

• 

  10: R44S

Side-chain optimization of SCWRL models over five iterations minimise. Green: the input model. </figure>

<figure id="fig:minimise_foldx_mutations">

• 

  1: H60R

• 

  2: V172I

• 

  3: E111K

• 

  4: L197P

• 

  5: W209R

• 

  6: L470P

• 

  7: W312C

• 

  8: A384D

• 

  9: D443N

• 

  10: R44S

Side-chain optimization of FoldX models over five iterations minimise. Green: the input model. </figure>

Gromacs

Runtime analysis

To show the relationship between nsteps and runtime of 'mdrun', different nstep were chosen from 50 to 1000. Three different energy functions were selected:

 AMBER03 protein, nucleic AMBER94
 CHARMM27 all-atom force field (with CMAP)
 OPLS-AA/L all-atom force field

nstep=50

step=45
real    0m6.770s
user    0m12.000s
sys     0m0.970s

nstep=100

step=40
real    0m6.039s
user    0m10.690s
sys     0m0.850s

nstep=200

step=44
real    0m6.700s
user    0m11.640s
sys     0m1.010s

nstep=300

Step=90

real    0m12.573s
user    0m23.430s
sys     0m1.210s

nstep=400

Step=20

real    0m3.537s
user    0m5.670s
sys     0m0.730s

nstep=500

nstep=600

nstep=700

nstep=800

nstep=900

nstep=1000

step=61

real    0m8.915s
user    0m16.210s
sys     0m0.990s

nstep=1500

step=50
real    0m7.583s
user    0m13.250s
sys     0m1.000s

nstep=2000

step=29
real    0m4.637s
user    0m7.850s
sys     0m0.740s

nstep=2500

nstep=3000

nstep=5000

Mutations

Mutation 1

Energy                      Average   Err.Est.       RMSD  Tot-Drift
-------------------------------------------------------------------------------
Bond                        1624.88        820    5492.83   -5022.62  (kJ/mol)
Angle                       4289.66         76    402.822   -411.894  (kJ/mol)
Potential                  -45289.6       2900    16896.6   -18817.6  (kJ/mol)

References

<references/>